Mouse monoclonal anti tubulin

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom

Mouse monoclonal anti-tubulin is a laboratory product used for the detection and analysis of tubulin, a key structural protein found in the cytoskeleton of eukaryotic cells. This antibody specifically binds to tubulin, enabling its identification and quantification in various experimental settings.

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34 protocols using mouse monoclonal anti tubulin

Investigating ErbB2 and Related Signaling

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The antibodies used are as follows: monoclonal mouse anti-actin (691001; MP Biomedicals, Santa Ana, CA); polyclonal rabbit anti-ErbB2 (HER2/neu), rabbit anti-nucleolin (C23), rabbit anti-Erk2 and rabbit anti-Akt, monoclonal mouse anti-phosphotyrosine (pY20) and monoclonal mouse anti-GFP and mouse anti-nucleolin (sc-284; sc-13057; sc-154; sc-8312; sc-508; sc-9996; sc-8031, respectively; Santa Cruz Biotechnology, Dallas, TX); monoclonal mouse anti-tubulin (T7816; Sigma-Aldrich); polyclonal rabbit anti-phospho-ErbB2, rabbit anti-phospho-Akt, rabbit anti-phospho-Erk1/2 (2249; 4058; 9101, respectively; Cell Signaling Technology, Danvers, MA); and monoclonal mouse anti-ErbB2 (extracellular; L87), which was a gift from Prof. Y. Yarden, Weizmann Institute of Science, Israel.
The aptamer GroA (AS1411) and the inactive oligomer Cro, were purchased from IDT (Jerusalem, Israel) as unmodified desalted oligonucleotides, as previously described [38 (link)].

Wolfson E., Goldenberg M., Solomon S., Frishberg A, & Pinkas-Kramarski R. (2016). Nucleolin-binding by ErbB2 enhances tumorigenicity of ErbB2-positive breast cancer. Oncotarget, 7(40), 65320-65334.

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Immunofluorescence Analysis of Tubulin

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Promastigote cells were incubated for 72 h without RAD or with the IC₉₀ of RAD. After the incubation period, cells (1 × 10⁷ cells) were sedimented, washed twice with PBS and suspended in 1 ml of PBS. Aliquots of the suspension (2 × 10⁵ cells) were applied on microscopic slides and air-dried. After fixing the cells for 2 min in ice-cold methanol, the slides were air-dried for 20 min. Non-adherent cells were removed by gentle washing (0.1% Triton X-100 in PBS) followed by incubation in blocking solution (2% BSA, 0.1% Triton-X 100 in PBS). Slides were then incubated for 1 h with monoclonal mouse anti-tubulin (Sigma-Aldrich, München, Germany, 1:4000), washed thrice and then incubated for 1 h with anti-mouse Alexa 594 (Dianova, Hamburg, Germany, 1:250) and DAPI (Sigma-Aldrich, München, Germany, 1:25). After washing the slides thrice, Mowiol and coverslips were applied and the slides were left to dry for 24 h at 4 °C. Fluorescence microscopy was carried out on an EVOS FL Auto epifluorescence microscope (Life Technologies, Darmstadt, Germany).

Hombach-Barrigah A., Bartsch K., Smirlis D., Rosenqvist H., MacDonald A., Dingli F., Loew D., Späth G.F., Rachidi N., Wiese M, & Clos J. (2019). Leishmania donovani 90 kD Heat Shock Protein – Impact of Phosphosites on Parasite Fitness, Infectivity and Casein Kinase Affinity. Scientific Reports, 9, 5074.

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Antibodies Used for JAM-A Detection

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The murine monoclonal anti–JAM-A antibodies 1H2A9, J10.4, and JF3.1 were purified as described (Liu et al., 2000 (link); Mandell et al., 2004 (link)). Other antibodies were purchased as follows: polyclonal affinity-purified rabbit anti–JAM-A (Invitrogen), monoclonal mouse Rap2 and polyclonal affinity-purified rabbit ZO-2 (BD Transduction Laboratories, San Jose, CA), and polyclonal affinity-purified rabbit anti-afadin 02246, monoclonal mouse anti-tubulin, and polyclonal affinity-purified rabbit anti-actin (Sigma-Aldrich, St. Louis, MO). For immunoblots, horseradish peroxidase–conjugated secondary antibodies were used (Jackson ImmunoResearch Laboratories, West Grove, PA). For immunofluorescence studies, fluorescein isothiocyanate– and Alexa-conjugated antibodies (Invitrogen) were used.

Monteiro A.C., Luissint A.C., Sumagin R., Lai C., Vielmuth F., Wolf M.F., Laur O., Reiss K., Spindler V., Stehle T., Dermody T.S., Nusrat A, & Parkos C.A. (2014). Trans-dimerization of JAM-A regulates Rap2 and is mediated by a domain that is distinct from the cis-dimerization interface. Molecular Biology of the Cell, 25(10), 1574-1585.

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Detection of Secreted and Cellular Proteins

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For detection of secreted AGR3, conditioned media were collected after 48 h of cell culture in serum-free DMEM, centrifuged at 13,000 rpm for 10 min, followed by overnight precipitated with cold acetone (at 80% final concentration). For cellular proteins detection, cells were lysed in lysis buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na₃CO₄, 1% Nonidet P40) containing protease inhibitor cocktail and phosphatase inhibitor cocktail 2 (both Sigma-Aldrich). For detection of tyrosine-phosphorylated proteins, the aforementioned lysis buffer was supplemented with 100 µM sodium orthovanadate (Sigma). The primary antibodies used were as follows: Mouse monoclonal anti-tubulin (Sigma), goat monoclonal anti-actin (Santa Cruz Biotechnology), mouse monoclonal anti-GAPDH (Santa Cruz Biotechnology), in-house mouse monoclonal anti-AGR3 (8 (link),25 (link)), Py-Plus™-HRP mouse anti-phosphotyrosine (Invitrogen), rabbit monoclonal anti-c-Src and anti-phospho-c-Src (both Cell Signaling Technology), mouse monoclonal anti-GAPDH (clone 6C5) (Millipore), mouse monoclonal anti-VCP (p97) (BD Biosciences) and rabbit monoclonal anti-EsRalpha antibody (Abcam).

Obacz J., Sommerova L., Sicari D., Durech M., Avril T., Iuliano F., Pastorekova S., Hrstka R., Chevet E., Delom F, & Fessart D. (2019). Extracellular AGR3 regulates breast cancer cells migration via Src signaling. Oncology Letters, 18(5), 4449-4456.

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Western Blotting of Muscle Differentiation Markers

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Cells were lysed in modified RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% IGEPAL, protease and phosphatase inhibitors) and 10–30μg of total protein was loaded into 10% SDS-PAGE gels and transferred to PVDF membranes for Western blotting with the following primary antibodies and dilutions: mouse monoclonal anti-Pax7, 1:10; mouse monoclonal (F5D) anti-myogenin (Developmental Studies Hybridoma Bank, USA), 1:5; mouse monoclonal anti-tubulin, 1:10000; mouse monoclonal anti-HA HRP conjugated, 1:4000 (Sigma-Aldrich,USA); mouse monoclonal anti-HDAC2 [3F3], 1:5000; rabbit polyclonal ChiP-grade anti-HA tag, 1:10000; rabbit polyclonal anti-Nedd4, 1:10000; rabbit monoclonal anti-GFP E385, 1:5000 (Abcam, UK); mouse monoclonal anti-GAPDH (EMD-Millipore, USA), 1:10000; mouse monoclonal 9B11 myc-tag (Cell Signaling, USA), 1:1000; rabbit polyclonal anti-GST (gift from Dr. María Paz Marzolo, Santiago, Chile), 1:5000 and mouse monoclonal P4D1 anti-ubiquitin (Santa Cruz Biotechnology, USA), 1:500. As secondary antibodies HRP conjugated anti-mouse IgG and anti-rabbit IgG (Cell Signaling, USA) were used at 1:5000. HRP activity was detected using the SuperSignal West Dura Extended Duration Substrate (Thermo-Fisher Scientific, USA). When indicated, lysates/fractions were subjected to co-immunoprecipitation as described [16 (link)].

Bustos F., de la Vega E., Cabezas F., Thompson J., Cornelison D., Olwin B.B., Yates JR I.I.I, & Olguín H.C. (2015). NEDD4 REGULATES PAX7 LEVELS PROMOTING ACTIVATION OF THE DIFFERENTIATION PROGRAM IN SKELETAL MUSCLE PRECURSORS. Stem cells (Dayton, Ohio), 33(10), 3138-3151.

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Co-Immunoprecipitation of GFP Fusion Proteins

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To perform co-immunoprecipitation, transfected S2 cells were harvested and lysed on ice with lysis buffer (50 mM Tris-HCl/pH 7.4, 150 mM NaCl, 2 mM sodium vanadate, 10 mM sodium fluoride, 1% Triton X-100, 10% glycerol, 10 mM imidazole and 0.5 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged for 15 min at 20,000×g, 4°C and the resulting supernatant was incubated with Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, Paso Robles, California) conjugated to mouse monoclonal anti-GFP clone 20 (Sigma-Aldrich, St. Louis, Missouri) for 4 hr at 4°C. After washing once with lysis buffer, twice with lysis buffer containing 0.1% deoxycholate, and 3 times with lysis buffer lacking Triton X-100, the immunoprecipitates and total lysates were resolved on 7.5% SDS-PAGE gels followed by western blot analysis as previously described (Kim et al., 2013 (link)).
Primary antibodies used in western blotting were mouse monoclonal anti-tubulin (Sigma), mouse anti-Myc (Sigma-Aldrich), mouse monoclonal anti-Aequorea Victoria GFP JL-8 (Clontech), and rabbit anti-phospho-Tyr412-c-Abl (Cell Signaling, Beverly, Massachusetts).

Sterne G.R., Kim J.H, & Ye B. (2015). Dysregulated Dscam levels act through Abelson tyrosine kinase to enlarge presynaptic arbors. eLife, 4, e05196.

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Immunocytochemical Characterization of Cellular Proteins

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Cells were cultured on cover slips in a 12-well plate, fixed with ice-cold 1:1 acetone: Methanol solution for 30 s followed by 5-s washing with PBS, and blocked with 1% (w/v) BSA/1% (w/v) gelatin in PBS overnight in 4°C. Primary antibodies used in immunoblotting were mouse monoclonal anti-rabbit anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse monoclonal anti-SSAO 7–106 (AK 982/02), mouse anti-Syn6 (BD Transduction Laboratories, 610636), and goat polyclonal anti-mouse IgG Alexa488-conjugate (Molecular Probes, A-11001). Secondary antibodies were goat polyclonal anti-rabbit IgG Alexa488-conjugate (Thermo, A11034) and goat polyclonal anti-mouse IgG Alexa488-conjugate (Thermo, A31570). All antibodies were used at a dilution of 1:100. Cells were imaged with a Zeiss LSM 710. Images were processed with ZEN and Image J.

Yang H., Ralle M., Wolfgang M.J., Dhawan N., Burkhead J.L., Rodriguez S., Kaplan J.H., Wong G.W., Haughey N, & Lutsenko S. (2018). Copper-dependent amino oxidase 3 governs selection of metabolic fuels in adipocytes. PLoS Biology, 16(9), e2006519.

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Characterizing TRIM28 Expression and Function

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The U2OS, Wi38-VA13 and HEK293T cells were cultured in dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and cultured in an incubator containing 5% CO2 at 37 ℃. Human full-length TRIM28 cDNA was cloned into pDEST27 (Invitrogen) with GST tag and Plenti-HAFL-puro with HA-Flag (HAFL) double tags.
Cells were infected with lentivirus corresponding to shRNAs or overexpression vectors and selected with puromycin. Sense and antisense DNA oligos designed according to siRNA sequences were synthesized and cloned into pLKO.1-GFP vector. The siRNA sequences are: siTRIM28-1, 5′-CCUggCUCUgUUCUCUgUCCU-3′; siTRIM28-2, 5′-CUgAgACCAAACCUgUgCUUA-3′; siLuci, 5′-CUUACGCUGAGUACUUCGA -3′.
Antibodies used in this study include: rabbit polyclonal anti-TRIM28 (Abcam), rabbit polyclonal anti-Flag (Abmart), mouse monoclonal anti-tubulin (Sigma), rabbit polyclonal anti-GAPDH (Abmart), rabbit polyclonal anti-53BP1 (Novus), rabbit polyclonal anti-GST (Abmart), mouse monoclonal anti-PML (Millipore), rabbit polyclonal anti-Histone H3 (Abcam), rabbit polyclonal anti-H3K9me3 (Abcam) and rabbit polyclonal anti-SETDB1 (Proteintech).

Wang C., Songyang Z, & Huang Y. (2021). TRIM28 inhibits alternative lengthening of telomere phenotypes by protecting SETDB1 from degradation. Cell & Bioscience, 11, 149.

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Western Blot Analysis of Protein Expression

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Cells were lysed in RIPA lysis buffer (20 mM Tris–HCl, pH 7.4, 150 mM sodium chloride, 2 mM EDTA, 1% NP40, 1 mM DTT, 0.1 mM PMSF, Complete protease inhibitors (Roche)), 24 h after transfection. Proteins were separated by SDS-PAGE on a 12% gel, transferred to a nitrocellulose membrane and probed with primary and secondary antibodies. The following antibodies were used at a 1 : 1000 dilution: rabbit polyclonal anti-HA (Abcam), mouse monoclonal anti-myc (Cell Signaling), and mouse monoclonal anti-tubulin (Sigma). HRP-conjugated goat anti-rabbit IgG and donkey anti-mouse IgG (Jackson Immuno Research) were used as secondary antibodies at 1 : 5000. Reactive bands were revealed with Lumi-light and Lumi-light plus western blotting substrate (Roche). Luminescence was recorded using luminescent image analyser LAS 4000 (Fuji). Densitometric analysis was done using ImageJ. Readthrough was expressed as ratio of: intensity of MDH1x band (readthrough band)/intensity of MDH1x band + intensity of MDH band.

Hofhuis J., Schueren F., Nötzel C., Lingner T., Gärtner J., Jahn O, & Thoms S. (2016). The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code. Open Biology, 6(11), 160246.

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Plasmid Constructs and Antibodies

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PEGFP, pEGFP-RILP, pEGFP-RILPC33, pCDNA3_2XHA, pCDNA3_2XHARILP and pCDNA3_2XHA-RILP-C33 have been described previously.^{20-22 (link)} Rabbit polyclonal anti-HA (1:500, ab9110) and antigiantin (1:1000, ab24586) were from Abcam (Cambridge, UK). Mouse monoclonal antitubulin (1:500 for immunofluorescence analyses, 1:10000 for immunoblot analyses, T5168) was from Sigma-Aldrich. Rabbit anti-RILP polyclonal antibody (1:100) has been described previously.^{1 (link)} Secondary antibodies conjugated to fluorochromes (1:200) or horseradish peroxidase (HRP, 1:5000) were from Invitrogen (Carslbad, CA, USA) or GE Healthcare (Barrington, IL, USA).

Margiotta A., Progida C., Bakke O, & Bucci C. (2017). Characterization of the role of RILP in cell migration. European Journal of Histochemistry : EJH, 61(2), 2783.

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